P308: NOVEL COPY NUMBER ABERRATION-BASED CLASSIFICATION METHODS REFINE RISK ASSESSMENT IN PEDIATRIC B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA
Authors: G. Bedics, B. Egyed, L. Kotmayer, A. Benard-Slagter, K. de Groot, A. Bekő, L. L. Hegyi, S. Krizsán, G. Kriván, D. J. Erdélyi, G. Kovács, B. Kajtár, L. Pajor, Á. Vojcek, G. Ottóffy, A. Ujfalusi, C. Kiss, I. Szegedi, K. Bartyik, G. Péter, E. Sebestyén, Z. Jakab, A. Matolcsy, S. Savola, C. Bödör, D. Alpár
Journal: HemaSphere (2022)

Background: The genomic landscape of pediatric acute lymphoblastic leukemia (ALL) is heterogeneous with distinct copy number aberrations (CNAs) being detectable in vast majority of the patients. A subset of these alterations provides prognostic and/or predictive information; therefore, various CNA-based patient classifiers were introduced in the past. Aims: We applied a next-generation sequencing (NGS) based method to comprehensively screen for recurrent, disease-relevant CNAs in a cohort of Hungarian patients, allowing us to establish novel patient risk stratification approaches. Methods: Diagnostic bone marrow samples from 261 children with B-ALL and 22 matching samples drawn from 21 patients at first or second relapse were investigated by digital multiplex ligation-dependent probe amplification (digitalMLPA) using the ALL-specific D007 probemix. DigitalMLPA libraries were sequenced on an NGS platform. Whole chromosome gains and losses, as well as subchromosomal CNAs were simultaneously profiled. Survival rates were estimated using the Kaplan-Meier method and compared by log-rank tests in R version 4.1.2. Results: In total, 1,400 CNAs including numerical chromosomal aberrations and subchromosomal CNAs were detected in 93.5% of the diagnostic samples. On average, 5.36 CNAs were observed per patient with a mean of 2.45 subchromosomal alterations. Subtype-defining aberrations were identified in 36.0% of the patients with hyperdiploidy and iAMP21 detected in 32.2% and 3.8% of the cases, respectively. Numerous CNAs in disease-relevant genes responsible for cell cycle control, lymphoid development, signaling, or tumor suppression were identified. Considering all affected exons and an upstream region, 10 distinct patterns of IKZF1 deletion were observed. Unbiased co-segregation analysis revealed 15 positive and 3 negative correlations between the various CNAs, e.g. common co-occurrence of iAMP21 and CDKN2A/B deletion, and negative correlation between ETV6 and BTG1 deletions. Comparative analysis of diagnostic and matching relapse samples revealed characteristic temporal changes of copy number profiles with additional aberrations (65%), as well as both emerging and disappearing CNAs (30%) detected at relapse as compared with diagnosis. One patient did not show CNAs in the analyzed samples (5%). Prognostic subgroups determined based on cytogenetic findings were combined (i) with IKZF1del/IKZF1plus status and (ii) with CNA subgroups defined by a comprehensive digitalMLPA profiling. The combined genetic classification methods identified 3 and 4 patient subgroups, respectively, with significantly different 5-year progression-free survival rates. Summary/Conclusion: Comprehensive and highly optimized CNA profiling with digitalMLPA revealed subtype-defining gross-chromosomal changes and additional disease-relevant subchromosomal CNAs in a large cohort of Hungarian patients treated with ALL IC BFM protocols. Comparative scrutiny of matching diagnostic and relapse samples from 21 patients unveiled two different patterns of temporal evolution. Two novel risk stratification approaches have been established by combining cytogenetic data with digitalMLPA-based copy number profiling, with one of those laying more emphasis on CNA data than seen in previously introduced classifications, and the other one combining cytogenetic data with IKAROS status for the first time. DigitalMLPA offers a fast, reliable and standardizable DNA copy number analysis which is easily implementable in the diagnostic workflow of pediatric ALL.